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    IPRONIDAZOLE

    1.  EXPLANATION

         Ipronidazole (2-isopropyl-1-methyl-5-nitroimidazole) is used for
    the treatment of histomoniasis in turkeys and in swine dysentery. This
    compound has not been previously reviewed by the Joint FAO/WHO Expert
    Committee on Food Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         After an oral administration of 22.5 mg 2-14C-ipronidazole/kg
    bw to five bile cannulated female Charles River rats for four
    consecutive days, 31% of the total radioactivity was found in the
    feces and in the contents of the gastrointestinal tract, 34% was found
    in the bile, 27% in urine, and 1.4% in the carcass, liver and gut.
    Total recovery of radioactivity was 92% (Weiss  et al., 1981).

    2.1.2  Biotransformation

         Two major metabolites were isolated in the feces of rats and
    turkeys after an oral administration of ipronidazole. In turkeys the
    metabolite was identified as
    1-methyl-2-(2-hydroxyisopropyl)-5-nitroimadazole, which together with
    the unchanged drug accounted for about 40% of the excreted dose. In
    the aqueous extract of the feces of rats the metabolite was identified
    as 2,3-dihydro-2-(2-hydroxypropyl)-3-methyl-4-nitro-1H-imidazol-5-ol,
    which represented 12.4% of the administered dose. Although
    experimental details were not presented, it was stated that no
    nitro-reduced metabolite was isolated (Weiss  et al., 1981).
    Metabolism studies with the isomer of ipronidazole in which the 2 and
    5 positions are interchanged
    (5-isopropyl-1-methyl-2-nitro-1H-imidazole) in female beagle dogs
    revealed that after an oral administration of 50 mg 2-14C-labelled
    drug/kg bw, 50% of the total radioactivity was found in the urine.
    Together with a small amount of unchanged drug three main metabolites
    still containing the nitro group were isolated. One metabolite was
    identified as the isomer of one of the main metabolites of
    ipronidazole (1-methyl-2-(2-hydroxyisopropyl)-5-nitroimidazole). The
    formation of the two more polar metabolites also involved oxidative
    steps in the isopropyl chain leading to a diol and a carboxylic acid
    at the primary carbon (Assandri  et al., 1978).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

                                                                                  
    Species      Sex      Route       LD50            Reference
                                      (mg/kg bw)
                                                                                  

    Mouse        M&F      oral        970             Banzinger & Hane,
                          i.p.        600             1967.
                          s.c.        >400

    Rat          M&F      oral        920             ibid.
    (adult)

    Rat          M&F      oral        205             Pool & Steivis,
    (neonate)                                         1968.

    Rabbit       M&F      oral        960             Banzinger & Hane,
                                                      1967.
                                                                                  

    2.2.2.  Short-term studies

    2.2.2.1  Rats

         Diets containing ipronidazole at levels of 0, 20, 80 or 320 mg/kg
    bw/day were fed to groups of 5 Charles River rats of each sex for
    thirteen weeks. The rats were observed daily for clinical signs of
    toxicity and body weights were recorded weekly. Growth rate and food
    consumption were similar in all groups. Hematology and urine analysis
    values were not affected by ipronidazole treatment. Gross autopsy of
    the tissues did not reveal pathological changes due to drug treatment.
    Histopathological examinations of the tissues were not performed.
    Absolute kidney and liver weight of male animals from all treated
    groups were elevated; relative liver weights of all rats of the
    highest dose groups were also elevated (Sadek & Banzinger, 1968).

         A rat study using the same protocol as the previous study with 8
    Charles River rats per sex per group was performed. The rats were
    observed daily for signs of clinical toxicity and body weights were
    recorded weekly. The body weights were significantly decreased in
    males and females of the 320 mg/kg bw/day group. Mean food consumption
    was similar in all groups with the exception of the females
    administered 20 mg/kg bw/day. This group ate more food than the other
    female rats. Opthalmologic examinations, hematology, clinical
    chemistry and urine analysis values revealed no differences between
    control and treated groups. There was a decrease in absolute and
    relative ovarian weight, which was not accompanied by histological
    changes. Hepatocellular hypertrophy occurred in the high dose male
    group. The NOEL was 80 mg/kg bw/day. (Banarjee  et al., 1968).

    2.2.2.2  Dogs

         Ipronidazole was administered orally by capsule at levels of 0,
    20, 80 and 160 mg/kg bw/day 6 days a week for thirteen weeks to groups
    of two beagle dogs of each sex. The dogs were observed daily for
    clinical signs of toxicity and body weights were recorded weekly.
    Hematologic studies and urine analysis were performed at 2 to 4 week
    intervals. In the 160 mg/kg bw/day group two dogs died and another was
    moribund and sacrificed; the fourth dog was taken off treatment 7´
    weeks after the start of the study. All dogs of that group revealed
    loss of body weight, dehydration, salivation, lacrimation, muscular
    tremor, ataxia and dilatation of the pupils. Autopsy of the tissues
    revealed congestion of internal organs and hepatomegaly. Dogs of the
    80 mg/kg bw/day group showed mild lacrimation and salivation, slightly
    decreased body weights and increased liver weights. Histology of the
    liver revealed a mild granularity of the cytoplasm and an increase of
    mesenchymal cells in the biliary tract. For this study the NOEL was 20
    mg/kg bw/day (Sadek & Banzinger, 1968).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mice

         Diets containing 0, 20, 200 or 1000 ppm ipronidazole were fed to
    groups of 80 Charles River derived CD-1 mice of each sex (2 control
    groups were used) for 89 weeks for males and for 100 weeks for females
    (the time at which survival was 20%). The average consumption of
    ipronidazole based on body weight was 0, 3, 30 or 150 mg/kg bw/day.
    The mice were observed daily for clinical signs of toxicity and body
    weights were recorded weekly for the first 13 weeks and every two
    weeks thereafter. The group mean body weights and food consumption
    were comparable in treated and control groups. There were no
    significant differences in survival rates between treated and control
    animals.

         Gross autopsy did not show any treatment-related changes.
    However, in males and females of the highest dose group there was a
    significant increase (P<0.05) in hyperplasia and adenoma of the lung
    (Table 1).

         Although only adenomas and hyperplasia were significantly
    increased at the highest dose group, a slight increase in tumor
    multiplicity in both sexes and carcinoma of males was also seen at the
    highest dose administered. Although adenoma of the lung is a common
    tumor in this strain of mice, the high dose incidence exceeded the
    historical control range from 8 studies in the same laboratory (2/50
    to 10/59 for males and 2/59 to 11/59 for females). Historical control
    data were not available for hyperplasia but the increase over
    concurrent controls is significant (P<0.05). The NOEL in this study
    was 200 ppm, equal to 30 mg/kg bw/day based on hyperplastic and
    neoplastic lesions of the lung (Reno  et al., 1981).

    Table 1: Hyperplastic and neoplastic lesions of the lung of mice
             treated with irponidazole
                                                                                  

                    MALE                            FEMALE
                          drug in the diet (ppm)/number of animals

                     0     0    20    200    1000    0    0   20   200  2000
                                                                                  

                    78    79   77     78     78     79   78   80   79   79
                                                                                  

    Hyperplasia      8     8    3     11     36*     8    8    7   13   25*

    Adenoma          8     8    7     13     25*     6    5    3    4   20*

    Adenoma          0     2    1      2      7      0    0    1    1    6
    (multiple)

    Carcinoma        3     2    3      1      7      0    3    0    3    2

    Carcinoma        0     0    0      0      1      0    0    0    1    1
    (multiple)

    Adenoma and/or  11     9   10     13     27      6    8    3    6   21
    carcinoma
                                                                                  

    * P<0.05

         Additional ophthalmologic examinations were carried out in 13
    weeks intervals and at the end of the study. There were no findings
    that indicated any effect of ipronidazole treatment on the eye (Reno
     et al., 1981).

    2.2.3.2  Rats

         A combined carcinogenicity and chronic toxicity study was
    performed in rats. Diets containing 0, 20, 200 or 2000 ppm
    ipronidazole were fed to groups of 50 Sprague Dawley derived CFY rats
    of each sex per group for 109 weeks. The average consumption of
    ipronidazole based on body weight was 0, 1, 10 or 110 mg/kg bw/day.
    The rats were observed daily for clinical signs of toxicity and body
    weights were recorded weekly for the first 15 weeks and every two
    weeks thereafter. Comprehensive hematological examinations and
    clinical chemical tests were performed on 15 rats of each sex of each
    group; urine analysis was performed from pooled urine of 5 rats from
    each sex per group. These examinations were done on pretest, and at 6,
    13, 19 and 26 months. The body weights of male and female rats of the

    2000 ppm group were significantly decreased at 51 weeks. Due to the
    presence of large mammary tumors in some rats no statistical analysis
    was carried out thereafter; however, during the second year of the
    study group mean body weights of all treated groups were lower than
    those of the controls. Food consumption was similar to controls in the
    three dosed groups. Survival rates of male rats were similar in all
    groups; survival rates of low and high dosed female rats were
    decreased when compared to female controls.

         An increase in the incidence of neoplasms of the mammary gland
    was observed in high dosed females (incidence: 42/50, 32/50, 37/50,
    and 48/50 at 0, 20, 200 and 2000 ppm, respectively). The time of
    appearance of tumors was sooner in the 2000 ppm group; after 75 weeks
    the incidences were 7, 12, 10, and 18 at 0, 20, 200, and 2000 ppm
    respectively. In addition, the number of mammary tumors per tumor
    bearing rat was increased in the high dosed group compared to controls
    (mean number of mammary tumors per rat: 2.76, 2.72, 3.08, and 3.68 at
    0, 29, 200 and 2000 ppm, respectively). Historical control data
    obtained from three previous studies revealed a high incidence of
    benign mammary tumors in untreated females; the incidences were 22/43,
    31/44, and 29/47 in these studies. The results of this study indicate
    an effect of ipronidazole on mammary tumor formation in high dosed
    female rats. However, the high incidence of tumors in the control
    animals, which is common in this rat strain, precluded the
    determination of a definite NOEL.

         No clinical signs could be attributed to the administration of
    ipronidazole. Macroscopic and histological examinations of the tissues
    of treated animals did not reveal any additional differences that
    could be associated with ipronidazole intake. Changes were observed on
    certain hematological and clinical biochemistry parameters. These
    changes were slight and in view of the fact, that no such alterations
    were observed in a 13-week-study, using higher dosages, these findings
    were not considered to be of major biological significance (Brentnall
     et al., 1977).

    2.2.3.3  Dogs

         Diets containing 0, 20, 200 or 2000 ppm ipronidazole were fed to
    groups of 8 beagle dogs of each sex per group. An interim sacrifice of
    2 dogs of each sex per group at about one year was scheduled. The
    other dogs remained on this diet for 104 weeks. Dosage based on body
    weight, was equal to 0, 0.51, 5.4 or 62 mg/kg/ bw/day. The dogs were
    observed daily for clinical signs of toxicity and body weights were
    recorded weekly. Every three months physical and ophthalomological
    examinations and hematology, clinical biochemistry and urine analysis
    were carried out. The body weights of the male and female dogs of the
    2000 ppm group were significantly lower than in the controls at the
    end of the study. Although average food consumption was variable
    throughout the study, treated dogs tended to eat less than the

    controls. However, no significant dose-response relationship was
    established. With the exception of one male dog which died during
    study week 99, all animals survived the study. Clinical signs observed
    more frequently in the treated groups included dermatitis, otitis and
    salivation. Ophthalomologic examinations revealed no drug related
    effects. In both male and female dogs of the 2000 ppm group, alkaline
    phosphatase was significantly increased throughout the study; in
    addition, potassium levels were significantly decreased in males (with
    the exception of 24 months) in females, potassium levels revealed
    significant decreases only after 6 and 9 months. At sacrifice, fat
    depletion was noted in 10/11 animals of the high dose group, relative
    liver weights were significantly increased in males and females and
    relative lung weights were increased in the females in the high dose
    group. Histological examination showed an increase of
    intrahepatocellular granular pigment in the high dose group animals.
    In the lower dose groups no drug related effects were observed. In
    this study the NOEL was 200 ppm, equal to 5.4 mg/kg bw/day for dogs
    (Tucek, 1980).

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         In a three generation study CFY rats were fed diets containing 0,
    20, 200 or 2000 ppm ipronidazole. The average consumption of
    ipronidazole was equivalent to 0, 1, 10 or 100 mg/kg bw/day,
    respectively. Each group consisted of 20 female and 10 male rats which
    were fed these diets for 80 days after which the rats were mated.
    Mating performance, parturition, litter size and postnatal growth were
    unaffected by ipronidazole treatment. However, reduced growth in rats
    of both sexes and in pregnant dams was observed at a dietary
    concentration of 2000 ppm. The fertility indices were not affected,
    although degenerative changes in the testes (damage of the tubules,
    loss of spermatogenesis) occurred in one animal in all treated groups.
    The NOEL for this study was 200 pm, equivalent to 10 mg/kg bw/day
    (Dale, 1976).

    2.2.5  Special studies on cataractogenicity

         See under long-term/carcinogenicity studies.

    2.2.6  Special studies on embryotoxicity and teratogenicity

    2.2.6.1  Rats

         Pregnant Füllingsdorf albino rats were treated by stomach tube
    with doses of 0, 10 or 100 mg ipronidazole/kg bw/day from day 6 to day
    15 of gestation. Each group consisted of 39 animals. Control and
    treated animals were divided into two subgroups. Females from subgroup
    1 were sacrificed on day 20 of gestation and uteri and fetuses were

    examined microscopically. Females from the second subgroups were
    allowed to give birth and to keep their young through the lactation
    period. The young rats were then sacrificed on day 22 of age. No
    skeletal or visceral malformations were found. Conception rate and
    litter size, litter weight and resorption rates were similar in all
    groups. Survival and development of the offspring were not affected.
    Though the doses should have been higher in order to induce some overt
    maternal toxicity, such as weight loss, the NOEL in this study was 100
    mg/kg bw/day (Backes, 1976).

    2.2.6.2  Rabbits

         Pregnant Yellowsilver rabbits were treated by gavage with doses
    of 0, 1, 10 or 100 mg/kg bw/day from day 6 to day 18 of gestation.
    Each group consisted of 20 animals. On day 29 of gestation all animals
    were sacrificed and autopsied. No drug related skeletal and visceral
    malformations were observed in the fetuses. Toxic effects were found
    in the 100 mg group in the form of significantly delayed maternal
    weight development, sedation and a significant increase in the number
    of resorbed fetuses. The NOEL was 10 mg/kg bw/day. (Backes, 1977). 

    2.2.7  Special studies on genotoxicity

    
         Results of genotoxicity studies on ipronidazole
                                                                                  
                                      Concentration
    Test System     Test Object       of ipronidazole  Results    Reference
                                                                                  

    Ames Test (1)   S.typhimurium   0.1-0.5 mmol/1     Positive   Cantelli-Forti
                        TA 100                                     et al., 1983.

    Ames Test (1)   S.typhimurium   1-4 mg/ml          Positive   Schüpbach, 1976
                        TA 1530
                        TA 1532
                        TA 1964

    Fluctuation     Klebsiella      0.02-1.0 mmol/l    Positive   Voogd, 1981
    Test            pneumoniae
                    E.coli          0.02-1.0 mmol/l    Positive
                    Citrobacter     0.1-1.0 mmol/l     Positive
                    freundii

    Host Mediated   S.typhimurium   100 or 150         Positive   Schüpbach, 1976.
    Assay               TA1530        mg/kg bw
                        (Mouse)           p.o.
                                                                                  

    (1)without rat liver S9 fraction.
    
    Three other tests (Micronucleus Test, Dominant Lethal Test and Human
    Cytogenetics Assay) were conducted. The results of these tests were
    negative. However, the experimental designs of these studies were
    inadequate (Schüpbach, 1976).

    3.  COMMENTS

         Pharmacokinetic data from rats and data from studies on
    carcinogenicity in mice and rats, mutagenicity, embryotoxicity and
    teratogenicity in rats and rabbits and reproduction in rats, and from
    long-term and short-term studies in rats and dogs were considered.

         In the rat, after oral administration of 14C-labelled
    ipronidazole, 27% of the total radioactivity is excreted in urine, 34%
    in bile and 31% in the feces. Two hydroxylated metabolites still
    containing the 5-nitro group have been identified in the feces of
    turkeys and rats after oral administration of ipronidazole.

         Ipronidazole showed mutagenic properties in bacterial test
    systems. Because of the inadequate design of studies in mammalian test
    systems, the Committee could not properly evaluate the genotoxic
    potential of this drug.

         In a carcinogenicity study in Charles River CD1 mice in which
    ipronidazole was administered in the diet, a significant increase in
    the incidence of benign proliferative lesions (adenoma and
    hyperplasia) in the lung was observed at 1000 ppm in both sexes.
    Although this type of tumor is common in this strain of mice, the
    number of tumors also exceeded the range reported for the laboratory
    historical controls. In this study, the no-observed-effect level was
    200 ppm in the diet, equal to 30 mg/kg bw/day for mice.

         In a combined carcinogenicity and chronic toxicity study with
    Sprague Dawley CFY rats, dietary concentrations of ipronidazole of 0,
    20, 200 and 2000 ppm were used. Although a high incidence of mammary
    tumors was seen in all treated female groups (74-96%) and control
    female rats (84%), the incidence was higher in the high-dose group
    than in the controls. In addition, mammary tumors appeared sooner and
    the number of mammary tumors per tumor-bearing rat was higher in the
    females receiving high doses. The Committee concluded that the results
    of this study indicated an effect of ipronidazole on mammary tumor
    formation in female rats in the high-dose group. However, the high
    incidence of tumors in the control animals, which is common in this
    rat strain, precluded the determination of a definite
    no-observed-effect level. Although the results of this study showed
    changes in some hematological and clinical biochemical parameters,
    similar effects were not observed in a 90-day rat study in which
    higher doses of ipronidazole were used.

         In a chronic toxicity study in dogs, which received ipronidazole
    at 0, 20, 200 or 2000 ppm in the diet, decreased body weight, changes
    in clinical biochemical values, fat depletion and changes in liver and
    lung weight were observed in the high-dose group. The
    no-observed-effect-level was 200 ppm bw/day, equal to 5.4 mg/kg
    bw/day.

         Studies on embryotoxicity and teratogenicity in rats and rabbits
    did not reveal any effects at levels of 100 mg/kg bw/day for rats or
    10 mg/kg bw/day for rabbits, which represent the
    no-observed-effect-level for these studies.

         In a three-generation study in rats fed diets containing 0, 20,
    200 or 2000 ppm of ipronidazole, reduced growth was noted in the
    highest-dose group. The fertility indices were not affected, although
    degenerative changes in the testes occurred in one animal in each of
    the treated groups. The no-observed-effect-level for this study was
    200 ppm, equal to 10 mg/kg bw/day.

         In a 13-week study in which rats were given ipronidazole in the
    diet, a no-observed-effect-level of 80 mg/kg bw/day was established on
    the basis of hepatocellular hypertrophy.

         In a 13 week study in dogs in which the compound was administered
    in capsules, all of the dogs in the highest-dose group either died or
    were taken off treatment. Loss of body weight, dehydration and ataxia
    were the most prominent signs of toxicity. These clinical signs
    occurrred to a lesser degree in the middle-dose group. The
    no-observed-effect-level was 20 mg/kg bw/day in this study.

         The Committee was not able to establish an ADI because the rat
    carcinogenicity study was inadequate to determine a no-effect level
    for ipronidazole.

    5.  REFERENCES

    ASSANDRI, A., PERAZZI, A., ZERILLI, C.F., FERRARI, O., & MARTINELLI,
    E. (1978). Metabolism of 5-isopropyl-1-methyl-2-nitro-1H-imadozole.
     Drug Metab.Dispos., 6, 109-113.

    BACKES, G. (1976). Embryotoxic studies in the rat by oral
    administration of Ro-7-1554 (Ipronidazole). Unpublished report from
    Hoffmann-La Roche, Inc. Nutley, N.J., USA. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    BACKES, G. (1977). Embryotoxic studies in rabbits by oral
    administration of Ro-7-1554 (Ipronidazole). Unpublished report from
    Hoffmann-La Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    BANARJEE, B.N., HOWARD, D.J., WOODARD, M.W. & WOODARD, G. (1968).
    Ro-7-1554, safety evaluation by repeated oral administration to rats
    for 13 weeks. Unpublished report from Woodard Research Corporation,
    Herndon, Virginia, USA. Submitted to WHO by the US Coordinator of the
    Codex Alimentarius, US Department of Agriculture, Washington, D.C.

    BANZINGER, R. & HANE, D. (1967). Acute toxicity and dog tolerance
    testing of Ro-7-1554 (Histomonacide). Unpublished report from
    Hoffman-la Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    BRENTNALL, D.W., DALE, E., INGHAM, B. & FARR, M.J. (1977).
    Ipronidazole: Toxicity and tumorgenicity study in rats. Unpublished
    report from May & Baker, Ltd., Dagenham, Essex, England. Submitted to
    WHO by the US Coordinator of the Codex Alimentarius, US Department of
    Agriculture, Washington, D.C.

    CANTELLI-FORTI, G., ALCARDI, G., GUERRA, M.C., BARBARO, A.M. & BIAGI,
    G.L. (1983). Mutagenecity of a series of 25 nitroimadazoles and two
    nitrothiazoles in Salmonella typhimurium.  Terat.Carcinog.Mutag.,
    3, 51-63.

    DALE, M.J. (1976). Ipronidazole: study of effects on reproductive
    performance in rats for three generations. Unpublished report from May
    & Baker, Ltd., Dagenham, Essex, England. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    POOL, W. & STEIVIS, D. (1968). Acute neonatal toxicity of Ro-7-1554
    (Ipronidazole). Unpublished report from Hoffmann-La Roche, Inc.,
    Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex
    Alimentarius, US Department of Agriculture, Washington, D.C.

    RENO, F.E., DUDEK, L.E., KUNDZIUS, W., VOELKER, R.W. & MISTRETTA, L.M.
    (1981). Chronic toxicity study in mice, ipronidazole base (Ro-7-l554),
    final report. Unpublished report No. 131-128 from Hazleton
    Laboratories America Inc., Vienna, VA, USA. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    SADEK, S. & BANZINGER, R. (1968). Preclinical toxicity study of
    Ro-1554 in rats and dogs for thirteen weeks. Unpublished report from
    Hoffmann-La Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US
    Coordinator of the Codex Alimentarius, US Department of Agriculture,
    Washington, D.C.

    SCHUPBACH, M. (1976). Mutagen tests with Ro-7-1554 (Ipronidazole).
    Unpublished report from Hoffmann-La Roche, Inc., Nutley, N.J. USA.
    Submitted to WHO by the US Coordinator of the Codex Alimentarius, US
    Department of Agriculture, Washington, D.C.

    TUCEK, P.C. (1980). Chronic feeding study in dogs. Unpublished report
    from International Research and Development Corporation, Mattawan,
    Michigan. Submitted to WHO by US Coordinator of the Codex
    Alimentarius, US Department of Agriculture, Washington, D.C.

    WEISS, G., ROSE, N., DUKE, P. & WILLIAMS, T.H. (1981). A major rat
    faecal metabolite of irponidazole. Xenobiotic, 11, 207-215.

    VOOGD, C.E. (1981). On the mutagenicity of nitroimiedazoles. Mut.Res.,
    3, 243-277.


    See Also:
       Toxicological Abbreviations
       IPRONIDAZOLE (JECFA Evaluation)